The ability to modify microorganisms and cells of higher organisms by genetic engineering has made it possible to change certain of their specific characteristics and thereby alter the `natural` response of those organisms to various agents. Of particular interest are the responses of organisms to agents used because of their cytotoxic effect. For example, many compounds used in agriculture are directed to the killing of pests, weeds, or the like. Often these compounds can have a relatively long residence time or extended residue in the plants subjected to treatment by the compound.
In many situations it is desirable to differentiate the species to be retained from the species to be killed. For example, it is often necessary to selectively destroy weeds, yet have minimal impact on the economically valuable crop plants. For the most part, broad-spectrum herbicides have a sufficiently adverse effect on crops that their use must be limited to emergent use or careful postemergent application.
Some weed species are simply resistant to today's herbicides, increasing the importance of developing the production of effective herbicides. Moreover, as some weed species are controlled, competition is reduced for the remaining tenacious weed species. The development of genetically engineered herbicide-resistant crop plants could significantly improve weed-control by allowing fields to be treated with a single, concentrated application of the herbicide. Therefore, a one-step procedure could eliminate costly and perhaps ineffective repeated low-dosage herbicidal treatments, such as have been required in the past to avoid damaging conventional crops, but which may have also induced the emergence of spontaneous herbicide-resistant weeds. Herbicides with greater potency, broader weed spectrum and more rapid degradation after application would avoid the problem tic persistence of the chemical herbicide in the soil, such as typically results from frequently repeated applications, and which prevents rotation of crops sensitive to that herbicide.
Certain herbicides, while not used directly to control weeds in field crops, are used: as 'total vegetation control agents' to eliminate weeds entirely in certain right-of-way or industrial situations. However, these herbicides may be deposited by natural means, such as water run-off, onto areas where economically important crops are growing. As a result sensitive field crops may be killed or their growth seriously inhibited. It is therefore highly desirable to be able to modify viable cells to make them resistant to stressful cytotoxic agents.
Sulfonylureas, imidazolinones, and triazolopyrimidines are structurally diverse, agriculturally important herbicides. Their primary target site is the enzyme acetolactate synthase (ALS), which catalyses the first common step in a plant's biosynthetic pathway of valine, leucine and isoleucine. The molecular basis of sulfonylurea resistance has been extensively characterized. Mutant forms of certain microorganisms and upper level plants expressing altered ALS resistance to sulfonylureas have been identified, i.e., in Nicotiana tabacum (tobacco) by Chaleff & Mauvais, Science 224:1443-1445 (1984), and in Arabidopsis thaliana by Haughn and Sommerville, Mol. Gen. Genet. 204:266-271 (1986). Furthermore, single amino acid substitutions in the ALS gene have been shown to confer resistance to sulfonylureas in tobacco by Lee et al., EMBO J. 7:1241-1248 (1988), and in Arabidopsis by Haughn et al., Mol. Gen. Genet. 211:266-271 (1988). However, there have been recent reports, i.e., by Sarri et al., Plant Physiol. 93:55-61 (1990), and Hall et al., Plant Physiol. 93:962-966 (1990), indicating that fields, which had been treated repeatedly with sulfonylureas have shown an emergence of herbicide-resistant biotypes of weeds which had been previously, effectively controlled.
The imidazolinone herbicides, notably imazapyr, imazaquin and imazethapyr, are a particularly important class of herbicide As described in the "Herbicide Handbook of the Weed Science Society of America", 6th Ed., (1989), imazapyr (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyrid inecarboxylic acid), is a non-specific, broad-spectrum herbicide, whereas both imazaquin (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-quino linecarboxylic acid), and imazethapyr (2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl -3-pyridinecarboxylic acid) are crop-specific herbicides particularly suited to use with soybean or peanut crops. These herbicides offer low mammalian toxicity, permit low application rates to plant crops, and provide long duration broad-spectrum weed control in the treatment of agricultural crops. A crop made more resistant to imidazolinone herbicides offers a selective means to control and kill weeds without adversely affecting the crop plant.
Clearly then, an understanding of the method by which weeds become resistant to herbicides at the molecular level is essential to establishing a basis for the development of sound weed control programs. The molecular basis underlying the expression of imidazolinone-resistance had remained undetermined until the present invention.
U.S. Pat. No. 4,761,373, issued to Anderson, discloses the production of plants, plant tissues and plant seeds which are herbicide-resistant, particularly plants, plant tissues and plant seeds which exhibit resistance to such herbicides resulting from the expression of genes encoding herbicide resistant acetohydroxyacid synthase. In the patent, Anderson contemplated introducing the herbicide resistance into any agronomically important crop. However, until the present invention, neither the imidazolinone-resistant ALS gene, nor the molecular basis of the expressed activity were understood. The imidazolinone-resistance trait has proven to be transferable to other maize lines only by classical breeding techniques; but, until the current invention it has not been possible to transfer the resistance to other monocot or dicot species by genetic engineering.
Early herbicide-enzyme kinetics data by Schloss et al., in Nature 331:360-362 (1988), proposed that sulfonylureas, imidazolinones and trizolopyrimidines shared a common binding site on a bacterial ALS. However, additional studies by several inventors, including recent experiments by Saxena et al., Plant Physiol. 94:1111-1115 (1990) and Sathasivan et al., Nucleic Acids Res. 18:2188 (1990), have indicated that with the exception of a few cases, the mutant forms of ALS which were resistant to imidazolinone lacked cross-resistance to sulfonylureas. Therefore, identification of the mutation site(s) in the ALS gene which code for the mutant plant's imidazolinone resistance is of agricultural significance.
Furthermore, the mechanism of inhibition was shown to be dissimilar between the imidazolinone and sulfonylurea herbicides. Imidazolinones inhibit ALS activity by binding noncompetitively to a common site on the enzyme, as demonstrated by Shaner et al., Plant Physiol. 76:545-546 (1984). By comparison, sulfonylureas inhibit ALS activity by competition as described by La Rossa and Schloss in J. Biol. Chem. 259:8753-8757 (1984). Therefore, since the mechanism of action of imidazolinone appears to be different from that of sulfonylurea herbicides, understanding the molecular basis of imidazolinone resistance is of great interest.
There remains a long-felt need in the art for the isolation of a mutant ALS gene, which confers resistance to imidazolinone in higher plants, and which would provide an opportunity to introduce imidazolinone resistance into crop plants by genetic engineering. Imidazolinones, because of their broad-spectrum activity and low mammalian toxicity, are particularly suited as a type of herbicide to which genetically engineered resistance would be economically important in crop plants. The development of imidazolinone-resistant crops would provide a reliable and cost-effective alternative to conventional weed management programs.
By modifying crop plant cells by the introduction of a functional gene expressing the imidazolinone-resistant ALS enzyme, one can use imidazolinones, including imazaquin and imazapyr, or an analogous herbicide with a wide variety of crops at a concentration which ensures the substantially complete or complete removal of weeds, while leaving the crop relatively unaffected. In this manner, substantial economies can be achieved in that fertilizers and water may be more efficiently utilized, and the detrimental effects resulting from the presence of weeds avoided.